Proteins produced by expression of cellular DNA may be divided into three general groups: Cytoplasmic proteins, which remain wholly within the cell; extracellular or "soluble" proteins, which are secreted outside the cell; and membrane proteins, which become attached to the phospholipid bilayer of the cell membrane.
Different membrane proteins play major roles in a wide range of cell functions, such as anchoring cytoskeletal components, mediating cell-to-cell adhesion, transporting molecules into and out of the cell, receiving signals from hormones and other chemical transmitters, and many others.
Membrane proteins are bound to the plasma membrane in a variety of ways. Peripheral or "extrinsic" proteins do not penetrate the phospholipid bilayer but instead interact with the polar head groups on one surface of the bilayer or interact with other membrane proteins anchored directly to the bilayer. Integral or "intrinsic" proteins have a region which interacts with the hydrophobic core of the bilayer. Transmembrane proteins, such as cell surface antigens, cellular receptors, adhesion molecules and transport proteins, traverse the bilayer one or more times, exposing both cytoplasmic and extracellular regions or domains.
A further possibility for attachment to the plasma membrane is via covalent linkages between the lipid bilayer and the protein. In particular, a number of cell surface proteins are anchored to the cell membrane through a C-terminal, covalently attached glycosylated phosphatidylinositol moiety. See, e.g., Low et al., Trends Biochem. Sci., 11, 212-15 (1986). The glycosyl phosphatidylinositol linkage, or "PI" linkage, is believed to be the anchoring structure for more than two dozen specific membrane proteins, found in a wide distribution of species and cell types See, Ferguson et al., Ann. Rev. Biochem., 57, 285-320 (1988) (incorporated herein by reference). The biological functions of these PI-linked proteins are also highly diverse, including surface hydrolases, coat proteins, surface antigens and adhesion molecules.
The C-terminal amino acid residue of the PI-linked protein is attached to the membrane phosphatidylinositol moiety via an ethanolamine-phos-phodiester-glycan bridge. Ferguson et al., Science, . 239, 753-759 (1988); Low, Biochem. J., 244, 1-13 (1987) (incorporated herein by reference). The mechanism of attachment is thought to involve processing of a C-terminal hydrophobic sequence present on the precursor protein but eliminated prior to the PI anchoring of the mature protein. Ferguson et al., supra, Ann. Rev. Biochem., 57, at 301-304. The processed C-terminal segments are believed to be a signal for phosphatidylinositol attachment. In one study, DNA coding for the 37-amino acid C-terminal sequence of the PI-linked protein, decay accelerating factor (DAF), was fused to the 3' end of DNA coding for a normally secreted protein fragment of glycoprotein D (from herpes simplex virus-1), resulting in a PI-linked fusion protein. See, Caras et al., Science 238, 1280-1283 (1987). However, comparison of the C-terminal sequences of many precursors of PI-linked proteins has failed to reveal a consensus PI linkage signalling sequence. See, Low, supra, FIG. 3; Ferguson et al., supra, Ann. Rev. Biochem., 57, Table 3.
Although there has been a considerable amount of investigation of phosphatidylinositollinked proteins and the phosphatidylinositol linkage, there is a need for further characterization of specific PI linkages and for further investigation into the signal and mechanism for PI attachment to the cellular membrane, as well as the selective release of membrane bound proteins. These needs are addressed by the present invention, relating to the PI linkage structure of a PI-linked form of lymphocyte function-associated antigen 3 ("LFA-3"), and to applications of PI linkage signalling sequences derived from PI-linked LFA-3.
The PI linkage signalling sequences of the present invention, when linked in frame to the 3' end of DNA coding for a secreted polypeptide, or the secreted portion of a polypeptide, result on expression in a PI-linked form of the polypeptide. Using the DNA sequences and methods herein, novel chimeric proteins bearing a C-terminal phosphatidylinositol structure and numerous applications for such products are made possible.